Gold Nanoparticles Impinge on Nucleoli and the Stress Response in MCF7 Breast Cancer Cells

Cancer cells can take up gold nanoparticles of different morphologies. These particles interact with the plasma membrane and often travel to intracellular organelles. Among organelles, the nucleus is especially susceptible to the damage that is inflicted by gold nanoparticles. Located inside the nucleus, nucleoli are specialized compartments that transcribe ribosomal RNA genes, produce ribosomes and function as cellular stress sensors. Nucleoli are particularly prone to gold nanoparticle-induced injury. As such, small spherical gold nanoparticles and gold nanoflowers interfere with the transcription of ribosomal DNA. However, the underlying mechanisms are not fully understood. In this study, we examined the effects of gold nanoparticles on nucleolar proteins that are critical to ribosome biogenesis and other cellular functions. We show that B23/nucleophosmin, a nucleolar protein that is tightly linked to cancer, is significantly affected by gold nanoparticles. Furthermore, gold nanoparticles impinge on the cellular stress response, as they reduce the abundance of the molecular chaperone hsp70 and O-GlcNAc modified proteins in the nucleus and nucleoli. Together, our studies set the stage for the development of nanomedicines that target the nucleolus to eradicate proliferating cancer cells

Co-immunostaining of HuR and aquaporin-1 during the recovery from renal ischemia

After 30min ischemia and a 4-hour recovery period, a mouse kidney was processed for immunofluorescence staining according to our protocol. Part A shows the fluorescence signals obtained with antibodies against HuR (red) or aquaporin-1 (AQP1, green) and DAPI staining of the DNA (blue). Except for red blood cells (and probably some endothelial cells), no aquaporin-1 was detected in the glomerulus, while the water channel was readily detected in tubular structures. Part B focuses on the glomerulus, where HuR was concentrated in nuclei. The zoomed-in region (Zoom), marked as white square, reveals details of HuR and aquaporin-1 distribution in the glomerulus

Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

<br><div><p><b>Background.</b> Chaperones and their co-factors are components of a cellular network; they collaborate to maintain proteostasis under normal and harmful conditions. In particular, hsp70 family members and their co-chaperones are essential to repair damaged proteins. Co-chaperones are present in different subcellular compartments, where they modulate chaperone activities.</p><p><b>Methods and Results.</b> Our studies assessed the relationship between hsc70 and its co-factor HspBP1 in human cancer cells. HspBP1 promotes nucleotide exchange on hsc70, but has also chaperone-independent functions. We characterized the interplay between hsc70 and HspBP1 by quantitative confocal microscopy combined with automated image analyses and statistical evaluation. Stress and the recovery from insult changed significantly the subcellular distribution of hsc70, but had little effect on HspBP1. Single-cell measurements and regression analysis revealed that the links between the chaperone and its co-factor relied on (i) the physiological state of the cell and (ii) the subcellular compartment. As such, we identified a linear relationship and strong correlation between hsc70 and HspBP1 distribution in control and heat-shocked cells; this correlation changed in a compartment-specific fashion during the recovery from stress. Furthermore, we uncovered significant stress-induced changes in the colocalization between hsc70 and HspBP1 in the nucleus and cytoplasm.</p><p><b>Discussion.</b> Our quantitative approach defined novel properties of the co-chaperone HspBP1 as they relate to its interplay with hsc70. We propose that changes in cell physiology promote chaperone redistribution and thereby stimulate chaperone-independent functions of HspBP1.</p></div

Fluorescent immunostaining of kidney sections with antibodies against aquaporin-1 to monitor the effects of ischemia

<div><p>Control (sham) or ischemic mouse kidneys were paraffin-embedded and processed as described in our protocol. Aquaporin-1 was detected with AlexaFluor®488-conjugated secondary antibodies (green); nuclei were stained withDAPI (blue). Scale bar is 25 µm; L, lumen of tubule, R, red blood cells.</p><div><br></div></div><ul></ul

Quantitative analysis of the interplay between hsc70 and its co-chaperone HspBP1

Background. Chaperones and their co-factors are components of a cellular network; they collaborate to maintain proteostasis under normal and harmful conditions. In particular, hsp70 family members and their co-chaperones are essential to repair damaged proteins. Co-chaperones are present in different subcellular compartments, where they modulate chaperone activities. [...

Book chapter: Exploring Subcellular Organization and Function with Quantitative Fluorescence Microscopy

Exploring Subcellular Organization and Function with Quantitative Fluorescence Microscopy; by Mohamed Kodiha, Hicham Mahboubi, Ursula Stochaj (2014). Book Chapter in Microscopy: advances in scientific research and education, 6th Ed (Formatex)

Data in support of 5′AMP-activated protein kinase alpha regulates stress granule biogenesis

AbstractThis data article contains insights into the regulation of cytoplasmic stress granules (SGs) by 5′-AMP-activated kinase (AMPK). Our results verify the specific association of AMPK-α2, but not AMPK-α1, with SGs. We also provide validation data for the isoform-specific recruitment of the AMPK-α subunit to SGs using (i) different antibodies and (ii) a distinct cellular model system. In addition, we assess the SG association of the regulatory AMPK β- and γ-subunits. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in “5′AMP-activated protein kinase alpha regulates stress granule biogenesis” [1]

Pifithrin-µ Induces Stress Granule Formation, Regulates Cell Survival, and Rewires Cellular Signaling

(1) Background: Stress granules (SGs) are cytoplasmic protein-RNA condensates that assemble in response to various insults. SG production is driven by signaling pathways that are relevant to human disease. Compounds that modulate SG characteristics are therefore of clinical interest. Pifithrin-µ is a candidate anti-tumor agent that inhibits members of the hsp70 chaperone family. While hsp70s are required for granulostasis, the impact of pifithrin-µ on SG formation is unknown. (2) Methods: Using HeLa cells as model system, cell-based assays evaluated the effects of pifithrin-µ on cell viability. Quantitative Western blotting assessed cell signaling events and SG proteins. Confocal microscopy combined with quantitative image analyses examined multiple SG parameters. (3) Results: Pifithrin-µ induced bona fide SGs in the absence of exogenous stress. These SGs were dynamic; their properties were determined by the duration of pifithrin-µ treatment. The phosphorylation of eIF2α was mandatory to generate SGs upon pifithrin-µ exposure. Moreover, the formation of pifithrin-µ SGs was accompanied by profound changes in cell signaling. Pifithrin-µ reduced the activation of 5′-AMP-activated protein kinase, whereas the pro-survival protein kinase Akt was activated. Long-term pifithrin-µ treatment caused a marked loss of cell viability. (4) Conclusions: Our study identified stress-related changes in cellular homeostasis that are elicited by pifithrin-µ. These insights are important knowledge for the appropriate therapeutic use of pifithrin-µ and related compounds